Coexistence of emerging bacterial pathogens in Ixodes ricinus ticks in Serbia

The list of tick-borne pathogens is long, varied and includes viruses, bacteria, protozoa and nematodes. As all of these agents can exist in ticks, their co-infections have been previously reported. We studied co-infections of emerging bacterial pathogens (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Francisella tularensis) in Ixodes ricinus ticks in Serbia. Using PCR technique, we detected species-specific sequences, rrf-rrl rDNA intergenic spacer for B. burgdorferi s.l., p44/msp2 paralogs for A. phagocytophilum, and the 17 kDa lipoprotein gene, TUL4, for F. tularensis, respectively, in total DNA extracted from the ticks. Common infections with more than one pathogen were detected in 42 (28.8%) of 146 infected I. ricinus ticks. Co-infections with two pathogens were present in 39 (26.7%) of infected ticks. Simultaneous presence of A. phagocytophilum and different genospecies of B. burgdorferi s.l. complex was recorded in 16 ticks, co-infection with different B. burgdorferi s. l. genospecies was found in 15 ticks and eight ticks harbored mixed infections with F. tularensis and B. burgdorferi s.l. genospecies. Less common were triple pathogen species infections, detected in three ticks, one infected with A. phagocytophilum / B. burgdorferi s.s. / B. lusitaniae and two infected with F. tularensis / B. burgdorferi s.s. / B. lusitaniae. No mixed infections of A. phagocytophilum and F. tularensis were detected.     

Infections and coinfections of questing Ixodes ricinus ticks by emerging zoonotic pathogens in Western Switzerland

In Europe, Ixodes ricinus is the vector of many pathogens of medical and veterinary relevance, among them Borrelia burgdorferi sensu lato and tick-borne encephalitis virus, which have been the subject of numerous investigations. Less is known about the occurrence of emerging tick-borne pathogens like Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis," and Anaplasma phagocytophilum in questing ticks. In this study, questing nymph and adult I. ricinus ticks were collected at 11 sites located in Western Switzerland. A total of 1,476 ticks were analyzed individually for the simultaneous presence of B. burgdorferi sensu lato, Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis," and A. phagocytophilum. B. burgdorferi sensu lato, Rickettsia spp., and "Candidatus Neoehrlichia mikurensis" were detected in ticks at all sites with global prevalences of 22.5%, 10.2%, and 6.4%, respectively. Babesia- and A. phagocytophilum-infected ticks showed a more restricted geographic distribution, and their prevalences were lower (1.9% and 1.5%, respectively). Species rarely reported in Switzerland, like Borrelia spielmanii, Borrelia lusitaniae, and Rickettsia monacensis, were identified. Infections with more than one pathogenic species, involving mostly Borrelia spp. and Rickettsia helvetica, were detected in 19.6% of infected ticks. Globally, 34.2% of ticks were infected with at least one pathogen. The diversity of tick-borne pathogens detected in I. ricinus in this study and the frequency of coinfections underline the need to take them seriously into consideration when evaluating the risks of infection following a tick bite.     

Association of Borrelia garinii and B. valaisiana with songbirds in Slovakia

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.     

Anaplasma phagocytophilum in small mammals and ticks in northeast Florida

Human anaplasmosis is an emerging tick-borne disease in the United States, but few studies of the causative agent, Anaplasma phagocytophilum, have been conducted in southeastern states. The aim of this study was to determine if A. phagocytophilum is present in small mammals and ticks in northeast Florida. Polymerase chain reaction assays designed to amplify portions of the major surface protein 2 gene (p44), 16S rDNA, and groESL operons were used to test rodent blood and tick DNA samples for the presence of A. phagocytophilum. Positive samples were confirmed by DNA sequence analysis. Anaplasma phagocytophilum DNA was detected in less than 5% of cotton mice and 45% of cotton rats from two sites in northeast Florida. Anaplasma phagocytophilum DNA was also confirmed in 1.3% of host-seeking adult Ixodes scapularis tested and 2.7% of host-seeking adult Amblyomma americanum. This report describes the first DNA sequence data confirming strains of A. phagocytophilum in rodents and ticks in Florida. The DNA sequences of the msp2, 16S rDNA, and groESL gene fragments obtained in this study were highly similar to reference strains of human pathogenic strains of A. phagocytophilum. These findings suggest that A. phagocytophilum is present and established among some small mammal species in northeast Florida. Although the infection prevalence was low in the total number of ticks tested, the presence of A. phagocytophilum in two human biting tick species, one of which is a known competent vector, suggests that humans in this region may be at risk of granulocytic anaplasmosis caused by this pathogen.     

Prevalence of Borrelia burgdorferi sensu lato in ticks collected from migratory birds in Switzerland

The prevalence of ticks infected by Borrelia burgdorferi sensu lato on birds during their migrations was studied in Switzerland. A total of 1,270 birds captured at two sites were examined for tick infestation. Ixodes ricinus was the dominant tick species. Prevalences of tick infestation were 6% and 18.2% for birds migrating northward and southward, respectively. Borrelia valaisiana was the species detected most frequently in ticks, followed by Borrelia garinii and Borrelia lusitaniae. Among birds infested by infected ticks, 23% (6/26) were infested by B. lusitaniae-infected larvae. Migratory birds appear to be reservoir hosts for B. lusitaniae.     

A new species of parasitic mites of the genus Syringophiloidus Kethley 1970 (Acari: Syringophilidae) from the barn swallow Hirundo rustica Linnaeus, 1758

A new quill mite species Syringophiloidus hirundinis (Acari: Syringophilidae) is described from the Barn Swallow Hirundo rustica. The species Syringophiloidus hirundinis had a prevalence of 17.1% in the two outermost tail feathers (N = 208 adult Barn Swallows) during the breeding season of the Barn Swallow host. Intensity of infestation was 9.7 adult mites per pair of infested tail feathers. The sex ratio was highly biased towards females, with only 7.5% of all individuals being males (20:3).     

Ixodes scapularis salivary gland protein P11 facilitates migration of Anaplasma phagocytophilum from the tick gut to salivary glands

Ixodes ticks harbour several human pathogens belonging to the order Rickettsiales, including Anaplasma phagocytophilum, the agent of human anaplasmosis. When ticks feed on A. phagocytophilum-infected mice, the pathogen enters the ticks' gut. The bacteria then migrate from the gut to infect the salivary glands of the ticks and are transmitted to the next host via the saliva. The molecular mechanisms that enable the migration of A. phagocytophilum from the gut to the salivary glands are poorly understood. Here we show that a secreted tick protein, P11, is important in this process. We show that P11 enables A. phagocytophilum to infect tick haemocytes, which are required for the migration of A. phagocytophilum from the gut to the salivary glands. Silencing of p11 impaired the A. phagocytophilum infection of tick haemocytes in vivo and consequently decreased pathogen infection of the salivary glands. In vitro experiments showed that P11 could bind to A. phagocytophilum and thus facilitate its infection of tick cells. This report provides new insights into A. phagocytophilum infection of ticks and reveals new avenues to interrupt the life cycle of Anaplasma and related Rickettsial pathogens.     

Ethiological agents of rickettsiosis and anaplasmosis in ticks collected in Emilia-Romagna region (Italy) during 2008 and 2009

Ticks are the main vectors of rickettsiae of the spotted fever group, as well as of a variety of other Rickettsiales, including bacteria of the genus Anaplasma, that might cause diseases in humans and animals. Here we present the result of a survey for ticks and for tick-associated Rickettsiales in the Emilia Romagna region (Northern Italy). The study was focused on ticks collected from wild-hunted animals. Out of 392 ticks collected from these animals, 282 (72%) were identified as Ixodes ricinus, 110 (28%) as Dermacentor marginatus. The former was found on four vertebrate species, whereas the latter appeared more specific for wild boar. The presence of rickettsiae was demonstrated in 22.5% of I. ricinus (57/253) and in 29% of D. marginatus (32/110). Five ticks of the species I. ricinus were also positive for Anaplasma phagocytophilum (2%). In addition, we collected ticks by dragging in a natural park of the same region. All of the ticks captured by dragging were identified as I. ricinus. Thirty-six out of 200 analyzed ticks proved positive for Rickettsia monacensis and R. helvetica (16.5 and 1.5%, respectively). Our results highlight that that ticks present in wild areas, widely exploited for recreation and hunting in Emilia-Romagna, represent a risk for the transmission of spotted fevers and anaplasmosis to humans.     

Co-phylogenetic analysis of Anaplasma phagocytophilum and its vectors, Ixodes spp. ticks

The coevolutionary history of Ixodes spp. ticks, the obligately tick-transmitted bacterial pathogen Anaplasma phagocytophilum, and its various rodent reservoir hosts world-wide is not known. According to coevolution theory, the most recently evolved of tick-bacterial complexes could have difficulty maintaining A. phagocytophilum in nature, because transmissibility has not been efficiently maximized. This study was intended to examine the phylogeographic history of I. ricinus-subgroup ticks and A. phagocytophilum, provide an estimate for the date of the divergence of A. marginale and A. phagocytophilum, and evaluate whether there is correspondence between tick and Anaplasma spp. trees. Analysis of Ixodes spp. ticks showed a New World clade consisting of I. scapularis and I. pacificus, European I. ricinus as a sister group to this clade, and Asian I. persulcatus as basal. Of the three A. phagocytophilum genes evaluated, the most resolution was provided by the ankA gene. ankA sequences formed an Old World clade with eastern North America strains as a sister clade. California strains were highly diverse and did not form a clade. Base substitution rates were very comparable along both A. marginale and A. phagocytophilum lineages. Based on 16S rDNA analysis, maximum and minimum divergence times of A. phagocytophilum and A. marginale were calculated to be 78,296,703 and 43,415,708 years, respectively. If A. phagocytophilum did closely coevolve with specific I. ricinus-subgroup tick species, then A. phagocytophilum strains could have specialized on local tick species and optimized local infectivity in the Old World and eastern US. However, lack of absolute resolution of tick trees and conflicting prevalence data (with low prevalence in Asia and western North America) preclude us from inferring a tight coevolutionary relationship of tick species from this phylogeographic analysis.     

Factors driving the abundance of ixodes ricinus ticks and the prevalence of zoonotic I. ricinus-borne pathogens in natural foci

Environmental factors may drive tick ecology and therefore tick-borne pathogen (TBP) epidemiology, which determines the risk to animals and humans of becoming infected by TBPs. For this reason, the aim of this study was to analyze the influence of environmental factors on the abundance of immature-stage Ixodes ricinus ticks and on the prevalence of two zoonotic I. ricinus-borne pathogens in natural foci of endemicity. I. ricinus abundance was measured at nine sites in the northern Iberian Peninsula by dragging the vegetation with a cotton flannelette, and ungulate abundance was measured by means of dung counts. In addition to ungulate abundance, data on variables related to spatial location, climate, and soil were gathered from the study sites. I. ricinus adults, nymphs, and larvae were collected from the vegetation, and a representative subsample of I. ricinus nymphs from each study site was analyzed by PCR for the detection of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum DNA. Mean prevalences of these pathogens were 4.0% ± 1.8% and 20.5% ± 3.7%, respectively. Statistical analyses confirmed the influence of spatial factors, climate, and ungulate abundance on I. ricinus larva abundance, while nymph abundance was related only to climate. Interestingly, cattle abundance rather than deer abundance was the main driver of B. burgdorferi sensu lato and A. phagocytophilum prevalence in I. ricinus nymphs in the study sites, where both domestic and wild ungulates coexist. The increasing abundance of cattle seems to increase the risk of other hosts becoming infected by A. phagocytophilum, while reducing the risk of being infected by B. burgdorferi sensu lato. Controlling ticks in cattle in areas where they coexist with wild ungulates would be more effective for TBP control than reducing ungulate abundance.     

A comparison of Chryseobacterium indologenes pathogenicity to the soft tick Ornithodoros moubata and hard tick Ixodes ricinus

A yellow-pigmented Gram-negative bacterium, Chryseobacterium indologenes, was found in the gut contents of about 65% of soft ticks Ornithodoros moubata from a perishing laboratory colony. The isolated putative pathogen, C. indologenes, was susceptible to cotrimoxazol and addition of this antibiotic (Biseptol 480) to the blood meal significantly decreased the tick mortality rate. The artificial infection of healthy O. moubata by membrane feeding on blood contaminated with C. indologenes was lethal to all ticks at concentrations 10(6) bacteria/ml. On the contrary, a similar infection dose applied to the hard tick Ixodes ricinus by capillary feeding did not cause significant mortality. Examination of guts dissected from infected O. moubata and I. ricinus revealed that C. indologenes was exponentially multiplied in the soft tick but were completely cleared from the gut of the hard ticks within 1 day. In both tick species, C. indologenes were found to penetrate from the gut into the hemocoel. The phagocytic activity of hemocytes from both tick species was tested by intrahaemocoelic microinjection of C. indologenes and evaluated by indirect fluorescent microscopy using antibodies raised against whole bacteria. Hemocytes from both tick species displayed significant phagocytic activity against C. indologenes. All O. moubata injected with C. indologenes died within 3 days, whereas the increase of the mortality rate of I. ricinus was insignificant. Our results indicate that hard ticks possess much more efficient defense system against infection with C. indologenes than the soft ticks. Thus, C. indologenes infection has the potential to be a relevant comparative model for the study of tick immune reactions to transmitted pathogens.     

Autochthonous and migratory birds as a dispersion source for Ixodes ricinus in southern Italy

The present study was carried out in a protected wooded area, which is part of the Parco Regionale Gallipoli Cognato Piccole Dolomiti Lucane, one of the most important ecological reserves in southern Italy. From April 2010 to April 2011, 212 birds, comprising 22 species from 12 families, were captured and examined for ticks. A total of 75 (35.4?%) birds were found infested by ticks, with 451 ticks being collected. All ticks were identified as Ixodes ricinus, of which 241 (53.4?%) were larvae and 210 nymphs (46.6?%). The highest intensity of infestation was found in April 2010, when 117 ticks were retrieved on 25 birds. No ticks were found on birds (n?=?5) netted in December 2010. High infestation rates were recorded on blackbirds (Turdus merula) (90 %; 29 birds examined) and on mistle thrushes (Turdus viscivorus) (100?%; 2 birds examined). The highest intensity of infestation by larvae was found on wrens (5.6 larvae/bird) and by nymphs on mistle thrushes (11.5 nymphs/bird). Temperature and number of hours of light showed to influence the activity of larvae and nymphs. These data support the notion that birds may be responsible for the heterogeneous distribution of I. ricinus in Europe, thus playing a role in the epidemiology of certain tick-borne pathogens.     

A review of mites of the subfamily Picobiinae Johnston & Kethley, 1973 (Prostigmata: Syringophilidae) from North American birds

The fauna of ectoparasitic mites of the subfamily Picobiinae (Acari: Syringophilidae) associated with birds of the North America is revised. A new genus, Charadriineopicobia n. g. is proposed for two quill mite species, Ch. calidris n. sp. from Calidris alba (Pallas) (Charadriiformes: Scolopacidae) and Ch. leucophaeus (Skoracki, Hendricks & Spicer, 2010) n. comb. from Leucophaeus atricilla Linnaeus (Charadriiformes: Laridae). The new genus differs from the closely related Neopicobia Skoracki, 2011 by the presence of one pair of setae in pseudanal series and by clearly discernible chambers in each lateral branch of the peritremes, in both sexes. Additionally, a new species of Picobia Haller, 1878, P. hylocichlae n. sp., parasitising Hylocichla mustelina (Gmelin) (Passeriformes: Turdidae), is described. The species of picobiine mites presently recorded from North America are summarised.     

A paretic condition in an Anaplasma phagocytophilum infected roe deer calf

This paper describes a case of Anaplasma phagocytophilum infection in a roe deer (Capreolus capreolus) calf in Norway. The calf was found deserted, paretic, and heavily infested with Ixodes ricinus ticks. It was euthanized and investigated postmortem. Anaplasma phagocytophilum was detected in several tissues by polymerase chain reaction (PCR) and 16S rRNA sequence analyses. Analyses for Borrelia burgdorferi sensu lato and tick-borne encephalitis (TBE) virus infections were negative. This is the first report of a possible paretic condition in A. phagocytophilum infected roe deer.     

Borrelia Burgdorferi Sensu Lato in Ixodes Ricinus Ticks and Rodents in a Recreational Park in South-Western Ireland

Ixodes ricinus ticks infected with Borrelia burgdorferi sensu lato were numerous on the edges of paths and roads in a recreational park in south-western Ireland. The abundance of ticks at different sites was related to the presence of deer, but a negative relationship was shown between tick abundance and tick infection rates. This is thought to be due to the deposition of large numbers of uninfected ticks by deer, which are apparently not good reservoir hosts of B. burgdorferi s.l. Blood meal analysis only detected deer DNA in uninfected nymphs. Reservoir competent rodents, Apodemus sylvaticus and Clethrionomys glareolus, were abundant at all sites and a high proportion of captured specimens were infested with larval ticks. However, very few rodents were infected with B. burgdorferi s.l. and none of the unfed infected nymphs analysed for the identity of their larval blood meal had fed on rodents. The spirochaetes detected in I. ricinus in the study area may be poorly adapted to rodents or are not transmitted readily because of the absence of nymphal infestation. The majority of spirochaetes in these ticks were apparently acquired from non-rodent hosts, such as birds.     

Loss of Lyme disease spirochetes from Ixodes ricinus ticks feeding on European blackbirds.

To determine whether blackbirds (Turdus merula), the most abundant and most abundantly tick-infested ecotonal bird of Central Europe, may contribute to the transmission of the Lyme disease spirochete (Borrelia burgdorferi), we compared the infectivity to ticks of naturally as well as experimentally infected blackbirds and rodents. European blackbirds experience intense exposure to Ixodes ricinus ticks and to the pathogens that they transmit. In nature, subadult I. ricinus ticks found feeding on these birds generally contain no spirochetes, although infection is universal in those found on black-striped mice (Apodemus agrarius). Those found on yellow-necked mice (A. flavicollis) are less frequently infected. Ticks lose infection in the course of feeding on blackbirds and fail to infect them. Subadult I. ricinus ticks readily feed on blackbirds, black-striped mice, and jirds (Meriones unguiculatus), but engorge less fully on the bird than on the rodents. Although birds may burden human health by establishing new infestations of I. ricinus ticks, our observations indicate that particular birds may benefit health by locally diminishing transmission of the Lyme disease spirochete.     

Birds and their ticks in northwestern California: minimal contribution to Borrelia burgdorferi enzootiology

Birds and their attendant ticks were surveyed for infection with the Lyme disease spirochete Borrelia burgdorferi, in chaparral and woodland-grass habitats in northwestern California from March to July, 1998 to 1999. In total, 234 birds were captured and recaptured (15%); nearly 2.5 times more birds were captured in chaparral than in woodland-grass. Overall, 34 species representing 15 families were collected during this study; of these, 24 species were caught in chaparral, 19 in woodland-grass, and 9 in both vegetational types. The most frequently captured birds were sage sparrows (Amphispiza belli) in chaparral, and American robins (Turdus migratorius) and oak titmice (Baelophus inornatus) in woodland-grass. Birds hosted 35 Ixodes pacificus (15 larvae, 20 nymphs) and 9 Haemaphysalis leporispalustris (3 larvae, 5 nymphs, 1 adult) ticks, of which 32 were removed from chaparral birds and 12 from woodland birds. The prevalence of tick infestation was 13% (21/167) in chaparral and 5% (3/67) in woodland-grass, but the relative and mean tick intensities of 0.19 and 1.5 for chaparral birds, and 0.18 and 4.0 for woodland birds, respectively, did not differ significantly by habitat. Spirochetes were not detected in either bird-blood or tick-tissue samples when tested by culture, immunofluorescence, or Giemsa-staining. In contrast, over 90% (86/94) of western fence lizards (Sceloporus occidentalis) collected in June or July were infested with an average of 6.9 and 8.9 immature I. pacificus in chaparral and woodland-grass, respectively. We conclude that birds contribute little to the enzootiology of B. burgdorferi in chaparral and woodland-grass habitats in northwestern California because of their limited parasitism by tick vectors and lack of detectable spirochetemias.     

Diversity and seasonal patterns of ticks parasitizing wild birds in western Portugal

The diversity and abundance of questing ticks and ticks parasitizing birds was assessed during 1?year in two recreational forests in western Portugal, a suburban forest and an enclosed game area. The aim of this study was to assess the distribution and seasonality of tick species and to understand the role of bird species as hosts for ticks. Ixodes ricinus was the most abundant questing tick collected in the enclosed game area, whereas in the suburban forest, only three ticks were collected by blanket dragging. Tick species parasitizing birds included I. ricinus, I. frontalis, I. arboricola, I. acuminatus, Haemaphysalis punctata, Hyalomma marginatum and H. lusitanicum. This is the first record of I. arboricola in Portugal. Tick prevalence and intensity of infestation differed between study areas and was higher in birds from the game area where a large population of deer and wild boar may support tick populations. Ground and shrub dwelling bird species such as Turdus merula, Erithacus rubecula and Sylvia melanocephala were the most heavily parasitized by ticks, but the importance of different bird species as hosts of larvae and nymphs of I. ricinus and I. frontalis differed. Therefore, different bird species may contribute differently for tick population maintenance.     

Three new species of picobiine mites (Acari: Syringophilidae) parasitising African flycatchers (Aves: Muscicapidae)

Three new species of quill mites of the subfamily Picobiinae Johnston & Kethley, 1973 (Acari: Syringophilidae) are described from African flycatchers (Passeriformes: Muscicapidae): Picobia cichladusa n. sp. on Cichladusa arquata Peters and P. myrmecocichla n. sp. on Myrmecocichla arnotti (Tristram), both from Tanzania, and P. echo n. sp. on Cossypha heuglini Hartlaub from the Democratic Republic of the Congo.